A NEW PCR-RFLP METHOD FOR THE IDENTIFICATION OF PARENTAL AND HYBRIDOGENETIC WESTERN EUROPEAN WATER FROGS INCLUDING THE Pelophylax perezi-grafi SYSTEM Angélica Cuevas, Cécile Patrelle, François Ciavatti, Thomas Gendre, Patricia Sourrouille, Philippe Geniez, Paul Doniol-Valcroze & Pierre-André Crochet Corresponding authors: Angélica Cuevas, angelica.cuevas@evobio.eu; Paul Doniol-Valcroze, pauldoniol-valcroze@orange.fr. CMYC2 nuclear marker alignment. The restriction site for the enzyme used (Ssp I) is aligned with the sequences and its restriction site is indicated in lower case on the first sequence. Pelophylax_lessonae_ITA_AB9802951 GAGCAGTTCT CAGTGCATGG ATCAAGATTT ATCAATTGAA A--CTCCACC TATTAGCAGC AACAGCAGCA GTTCTGAGTC [ 80] Pelophylax_lessonae_ITA_AB980296 .......... .......... .......... .......... .--....... .......... .......... .......... [ 80] Pelophylax_lessonae_MF667674 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_lessonae_MF667673 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_lessonae_MF094359 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_lessonae_MF094357 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_lessonae_MF094356 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_perezi_MF667661 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_ridibundus_TUR_AB980346 .......... .......... .......... .......... .--....... .......... .......... .......... [ 80] Pelophylax_ridibundus_TUR_AB980345 .......... .......... .......... .......... .CT....... .......... .......... .......... [ 80] Pelophylax_ridibundus_MF667672 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_ridibundus_MF667671 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_ridibundus_MF667670 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_ridibundus_MF667668 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_ridibundus_MF667666 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_ridibundus_Bed_MF667657 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_ridibundus_Bed_MF667658 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_ridibundus_Bed_MF667659 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_ridibundus_Bed_MF667660 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .....T.... .......... [ 80] Pelophylax_ridibundus_GRE_MF667663 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Pelophylax_ridibundus_GRE_MF667664 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNN....... .......... .......... .......... [ 80] Ssp I AATATT Pelophylax_lessonae_ITA_AB9802951 TGGTAAGTAC CAGTCAACTT CGTTT-ATTG GGTTTTT--- ATCATTGTAG aatattTTTA TGTTCATGGG GGAAGGTTAG [160] Pelophylax_lessonae_ITA_AB980296 .......... .......... T....-.... .......--- .......... .......... .......... .......... [160] Pelophylax_lessonae_MF667674 .......... .......... .....T.... .......--- .......... .......... .......... .......... [160] Pelophylax_lessonae_MF667673 .......... .......... .....T.... .......--- .......... .......... .......... .......... [160] Pelophylax_lessonae_MF094359 .......... .......... .....T.... .......--- .......... .........G ......C... .......... [160] Pelophylax_lessonae_MF094357 .......... .......... .....T.... .......--- .......... .......... .......... .......... [160] Pelophylax_lessonae_MF094356 .......... .......... .....T.... .......--- .......... .......... .......... .......... [160] Pelophylax_perezi_MF667661 .......... .......... A....T.... .......KTT .......... ..GG...... G......... ....T..... [160] Pelophylax_ridibundus_TUR_AB980346 .......... .......... A....T.... .......--- ...CA.A... ..GG...... .......... ...C.T.... [160] Pelophylax_ridibundus_TUR_AB980345 .......... .......... A....T.... .......--- ...CG.A... ..GG...... .......... .....T.... [160] Pelophylax_ridibundus_MF667672 .......... .......... A....T.... ..C....--- ...CG.A... ..GG...... .......... .....T.... [160] Pelophylax_ridibundus_MF667671 .......... .......... A....T.... ..C....--- ...CG.A... ..GG...... .......... .....T.... [160] Pelophylax_ridibundus_MF667670 .......... .......... A....T.... ..C....--- ...CG.A... ..GG...... .......... .....T.... [160] Pelophylax_ridibundus_MF667668 .......... .......... A....T.... .......--- ...CA.A... ..GG...... .......... .....T.... [160] Pelophylax_ridibundus_MF667666 .......... .......... A....T.... .......--- ...CG.A... ..GG...... .......... .....T.... [160] Pelophylax_ridibundus_Bed_MF667657 .......... .......... A....T.... .......--- ...CG.A... ..GG...... .......... .....T.... [160] Pelophylax_ridibundus_Bed_MF667658 .......... .......... A....T.... .......--- ...CA.A... ..GG...... .......... ...C.T.... [160] Pelophylax_ridibundus_Bed_MF667659 .......... .......... A....T.... .......--- ...CA.A... ..GG...... .......... .....T.... [160] Pelophylax_ridibundus_Bed_MF667660 .......... .......... A....T.... .......--- ...CA.A... ..GG...... .......A.. .....T.... [160] Pelophylax_ridibundus_GRE_MF667663 .........A -------... A....T.... .T.....--- ....G.T... ..G....... ......C... .....T.... [160] Pelophylax_ridibundus_GRE_MF667664 .........A -------... A....T.... .T.....--- ....G.T... ..GG...... ......C... .....T.... [160] Pelophylax_lessonae_ITA_AB9802951 ATGGCTTT-A TGTGATCTTT GAAGGCTTTG AGAAATGTT- AGGCTTTACG TGATCTTTAA AGGCTTTGAC TTTTCCTCCA [240] Pelophylax_lessonae_ITA_AB980296 ........-. .......... .......... .........- .......... .......... .......... .......... [240] Pelophylax_lessonae_MF667674 ........-. .......... .......... .........- .......... .......... .......... .......... [240] Pelophylax_lessonae_MF667673 ........-. .......... .......... .........- .......... .......... .......... AC........ [240] Pelophylax_lessonae_MF094359 ........-. .......... .......... .........- .......... .......... .......... .......... [240] Pelophylax_lessonae_MF094357 ........-. .......... .......... .........- .......... .......... .......... .......... [240] Pelophylax_lessonae_MF094356 ........-. .......... .......... .........- .......... .......... .......... AC........ [240] Pelophylax_perezi_MF667661 ........-. .......... .......... .........- ...T....Y. .......... .......... .......... [240] Pelophylax_ridibundus_TUR_AB980346 ........T. .......... .......... .........- .......... .......... .......... ...G...... [240] Pelophylax_ridibundus_TUR_AB980345 ........T. .......... .......... .........T .......... .......... .......... .......... [240] Pelophylax_ridibundus_MF667672 ........T. .......... .......... .........- .......... .......... .......... .......... [240] Pelophylax_ridibundus_MF667671 ........T. .......... .......... .........- .......... .......... .......... .......... [240] Pelophylax_ridibundus_MF667670 ........T. .......... .......... .........- .......... .......... .......... .......... [240] Pelophylax_ridibundus_MF667668 ........T. .......... .......... .........- .......... .......... .......... ...G...... [240] Pelophylax_ridibundus_MF667666 ........T. .......... .......... .........T .......... .......... .......... .......... [240] Pelophylax_ridibundus_Bed_MF667657 T.......T. .......... .......... .........- .......... .......... .......... ...G...... [240] Pelophylax_ridibundus_Bed_MF667658 ........T. .......... .......... .........- .......... .......... .......... ...G...... [240] Pelophylax_ridibundus_Bed_MF667659 T.......T. .......... .......... .........- .......... .......... .......... ...G...... [240] Pelophylax_ridibundus_Bed_MF667660 ........T. .......... .......... .........- .......... .......... .......... ...G...... [240] Pelophylax_ridibundus_GRE_MF667663 ........-. .......... .........C .........- ........T. .......... .......... .......... [240] Pelophylax_ridibundus_GRE_MF667664 ........-. .......... .........C .........- ........T. .......... .......... .......... [240] Pelophylax_lessonae_ITA_AB9802951 CGTAGAAGTC TTT [253] Pelophylax_lessonae_ITA_AB980296 .......... ... [253] Pelophylax_lessonae_MF667674 .......... ... [253] Pelophylax_lessonae_MF667673 .......... ... [253] Pelophylax_lessonae_MF094359 .......... ... [253] Pelophylax_lessonae_MF094357 .......... ... [253] Pelophylax_lessonae_MF094356 .......... ... [253] Pelophylax_perezi_MF667661 T......... ... [253] Pelophylax_ridibundus_TUR_AB980346 T.......G. ... [253] Pelophylax_ridibundus_TUR_AB980345 T.......G. ... [253] Pelophylax_ridibundus_MF667672 .......... ... [253] Pelophylax_ridibundus_MF667671 .......... ... [253] Pelophylax_ridibundus_MF667670 T......... ... [253] Pelophylax_ridibundus_MF667668 T.......G. ... [253] Pelophylax_ridibundus_MF667666 T.......G. ... [253] Pelophylax_ridibundus_Bed_MF667657 T.......G. ... [253] Pelophylax_ridibundus_Bed_MF667658 T.......G. ... [253] Pelophylax_ridibundus_Bed_MF667659 T.......G. ... [253] Pelophylax_ridibundus_Bed_MF667660 T.......G. ... [253] Pelophylax_ridibundus_GRE_MF667663 T.......G. ... [253] Pelophylax_ridibundus_GRE_MF667664 T.......G. ... [253]